Production of Volatile Moth Sex Pheromones in Transgenic Nicotiana benthamiana Plants

Abstract

Plant-based bioproduction of insect sex pheromones has been proposed as an innovative strategy to increase the sustainability of pest control in agriculture. Here, we describe the engineering of transgenic plants producing (Z)-11-hexadecenol (Z11-16OH) and (Z)-11-hexadecenyl acetate (Z11-16OAc), two main volatile components in many Lepidoptera sex pheromone blends. We assembled multigene DNA constructs encoding the pheromone biosynthetic pathway and stably transformed them into Nicotiana benthamiana plants. The constructs contained the Amyelois transitella AtrΔ11 desaturase gene, the Helicoverpa armigera fatty acyl reductase HarFAR gene, and the Euonymus alatus diacylglycerol acetyltransferase EaDAct gene in different configurations. All the pheromone-producing plants showed dwarf phenotypes, the severity of which correlated with pheromone levels. All but one of the recovered lines produced high levels of Z11-16OH, but very low levels of Z11-16OAc, probably as a result of recurrent truncations at the level of the EaDAct gene. Only one plant line (SxPv1.2) was recovered that harboured an intact pheromone pathway and which produced moderate levels of Z11-16OAc (11.8 μg g-1 FW) and high levels of Z11-16OH (111.4 μg g-1). Z11-16OAc production was accompanied in SxPv1.2 by a partial recovery of the dwarf phenotype. SxPv1.2 was used to estimate the rates of volatile pheromone release, which resulted in 8.48 ng g-1 FW per day for Z11-16OH and 9.44 ng g-1 FW per day for Z11-16OAc. Our results suggest that pheromone release acts as a limiting factor in pheromone biodispenser strategies and establish a roadmap for biotechnological improvements.

Tunable control of insect pheromone biosynthesis inNicotiana benthamiana

“…However, while the pheromones of some species can be cheaply manufactured by synthetic chemistry, the pheromones of many insect species have complex structures requiring stereoselective steps making them difficult and expensive to produce (Petkevicius et al, 2020). To provide proof-of-concepts for the biological synthesis of pheromone components, several studies have successfully produced Lepidopteran sex pheromones in heterologous systems including yeast (Hagström et al, 2013;Holkenbrink et al, 2020;Konrad et al, 2017;Petkevicius et al, 2020) and N. benthamiana (Ding et al, 2014;Mateos-Fernández et al, 2021;Xia et al, 2022Xia et al, , 2020. In the latter, both transient agroinfiltration and stable transgenic approaches have been trialed, though retardation of growth was observed in the best-producing transgenic lines (Mateos-Fernández et al, 2021).…”

“…The genes encoding moth acetyltransferases involved in pheromone biosynthesis have not yet been identified and previous studies have employed either a plant enzyme, EaDAcT (Ding et al, 2014;Mateos-Fernández et al, 2021) or the yeast enzyme ScATF1 (Ding et al, 2016;Xia et al, 2022), with the latter producing more acetate. When comparing the activities of these enzymes we observed differences in the levels of product obtained from two yeast enzymes, ScATF1 and SpATf1-2, which differ at only one amino acid (Figure 1) Inducible gene expression systems are essential tools in metabolic engineering.…”

“…Standards, extraction methods and analysis of pheromone compounds were as previously described (Mateos-Fernández et al, 2021). Briefly, synthetic samples of Z11-16OH were obtained as described by Zarbin et al (2007) and purified by column chromatography using silica gel and a mixture of hexane : Et2O (9 : 1 to 8 : 2) as an eluent.…”

“…Indeed, a major advantage of biological manufacturing is the ability to produce complex molecules, including those for which chemical synthesis has proven difficult or commercially non-viable due to the requirement for multiple stereoselective steps (Cravens et al, 2019). For example, there is growing interest in the biological production of insect sex pheromones (many of which are stereochemically complex) for species-specific control of agricultural pests (Ding et al, 2014; Holkenbrink et al, 2020; Mateos Fernández et al, 2022; Mateos-Fernández et al, 2021; Xia et al, 2021).…”

Reconstitution of monoterpene indole alkaloid biosynthesis in genome engineered Nicotiana benthamiana

“…However, several studies have reported the accumulation of unintended side products, presumably produced by the off-target activities of endogenous N. benthamiana enzymes such as oxidases and glycosylases [8][9][10][11][12][13][14][15][16][17][18] . Although the activity of endogenous enzymes has been exploited in some studies to produce novel molecules 8 or to compensate for the lack of a known enzyme 19 , derivatization of molecules for the most part is a disadvantage, reducing the potential purity and yield of the target compound.…”